Immunoassays are a type of ligand-binding assay and are widely used to determine the presence and quantities of analytes (i.e., substances or chemical constituents of a sample which are being detected). Agglutination immunoassays are a type of immunoassay in which the immunochemical reaction results in clumping of particulates such as red blood cells or polymeric latex particles. The use of immunochemical reactions as a means of causing agglutination has found application in the determination of many analytes, as described in: Nichols, W. S. and R. M. Nakamura, "Agglutination and Agglutination Inhibition Assays", Laboratory Manual of Clinical Immunology (Rose et al., ed.) 49-56 (1985).
For example, blood typing is performed by agglutination assays in which reagent antibody is added; red blood cells in the sample clump as a result of interaction between the added reagent antibody and antigens on the cell surfaces. Hemagglutination tests are immunoassays which use specially treated red blood cels. Such tests have been used for detecting antibodies and antigens, such as rubella antibody, rheumatoid factor, hepatitis antibody, hepatitis antigen and pregnancy hormone.
Reagents used in hemagglutination assays can be red blood cells (erythrocytes) which have antigens or antibodies bound to their surfaces. Red blood cells can be stabilized by cross linking or by treatment with tanning agents. See, for example, processes such as those taught in U.S. Pat. Nos. 4,403,037 and 4,587,222. For example, U.S. Pat. No. 4,403,037 describes preparation of antigen-coated erythrocytes with a cross-linking agent for the dual purpose of stabilizing the coated erythrocytes and reducing their hemagglutinating activity. Reduction of hemagglutinating activity is described as preventing the antigen-coated erythrocytes from undergoing spontaneous agglutination in the absence of antibody specific to red-blood-cell-bound hemagglutinating antigen. U.S. Pat. No. 4,587,222 describes a reagent containing red blood cells and soluble antibodies, as well as a process for making the reagents which involves subjecting its components to treatment with aldehydes or tanning agents.
Processes such as these can increase the useful life of the reagent red cells, but such chemical treatment also converts the flexible cell membrane to a rigid membrane and alters the surface properties of the cells. In some cases these changes adversely affect the specificity of the reagent in the assay.
For these reasons, polymeric latex particles have been used instead of red blood cells in some agglutination assays, such as in the agglutination assay for rheumatoid factor described in U.S. Pat. No. 4,547,466, which describes a method of preparing latex particles having immune complexes attached to their surfaces, and use of such particles.
However, reagent red blood cells and reagent polymeric particles both have the disadvantage of being likely to agglutinate even in the absence of the analyte being determined. In the case of reagent red blood cells, nonspecific agglutination is common and results from the presence of blood group antibodies and heterophile antibodies in the sample. In the case of polymeric particles, non-specific agglutination results from non-specific adsorption of proteins and other molecules in the sample to the particles.
Before presently-available methods based on agglutination can be carried out, it is generally necessary to remove the red blood cells from the sample. One exception to this is seen in blood-typing analyses, for which this is not necessary. Removal of red blood cells is not only an extra step in the procedure, but also one which may remove or alter the reactivity of the analyte being determined. U.S. Pat. No. 4,594,327 describes an immunochromatographic method for determination of an analyte in whole blood, in which two functions are combined in one step: separation of interfering cells (e.g., red blood cells) through binding to a binding agent and determination of the analyte.
U.S. Pat. Nos. 4,578,360 and 4,529,712 describe materials designed for use in immunoassay of antigens or antibodies; methods in which they are used are not agglutination immunoassays, but rely on other techniques of detecting analyte. In U.S. Pat. No. 4,578,360, Smith describes a mixed binding reagent (MBR) containing an antigen-binding site and a label-binding site; the reagent is described as normally consisting of two antibodies. In the method described, presence of an analyte (e.g., an antigen) is determined by mixing an analyte-containing sample with the MBR and a labelled substance and determining the quantity of labelled substance bound to the label-binding site of the MBR. In U.S. Pat. No. 4,529,712, heterobifunctional reagents are described for use in conjugating substances (e.g., antigens, antibodies) to membranes of cells or liposomes, which can then be used in hemolytic or immunocytoadherence assays.
Results of agglutination assays have generally been assessed visually, as described by Nichols and Nakamura. Nonvisual methods can also be used in some cases to detect agglutination. For example, U.S. Pat. Nos. 4,398,894; 4,436,827; and 4,554,257 describe nonvisual methods. These methods can be used for measuring hemagglutination assays.